Download Changes in the blood composition of some anurans

Acta Herpetologica 6(2): 137-147, 2011
Changes in the blood composition of some anurans
Çiğdem Gül*, Murat Tosunoğlu, Didem Erdoğan, Dilşah Özdamar
Çanakkale Onsekiz Mart University, Faculty of Sciences and Arts, Department of Biology, Zoology Section, Tr-17100 Çanakkale-Turkey. * Correspondig author. E-mail: [email protected]
Submitted on: 2010, 23th December; revised on: 2011, 28th April; accepted on: 2011, 10th May.
Abstract. We examined some hematological parameters (red blood cell count, white blood cell count, haemoglobin concentration, hematocrit value, mean cell volume,
mean cell haemoglobin, mean cell haemoglobin concentration and plasma total protein) on five anuran species of terrestrial (Pseudepidalea viridis, Pelobates syriacus and
Hyla arborea), semi-aquatic (Rana dalmatina) and aquatic (Pelophylax ridibundus)
nature from Çanakkale, Turkey. Differences between males and females in terms of
haemoglobin, hematocrit and mean cell volume in P. viridis were statistically significant. The RBC count was higher in terrestrial and aquatic species than in semi-aquatic
species. Haemoglobin concentration, hematocrit value, MCV and MHC were higher
in terrestrial species than in semi-aquatic and aquatic species. The MCHC values were
all similar to each other. The plasma total protein was higher in terrestrial species than
in aquatic species. To sum up, variations were detected in the some hematological
parameters under examination among the anuran species.
Keywords. Anura, some hematological parameters, terrestrial, semi-aquatic, aquatic.
INTRODUCTION
Amphibia and Reptilia species were affected by negative conditions of pollution in the
environmental condition and destruction of biotopes. Blood parameters of Amphibia species were particularly affected by the negative environmental conditions. Amphibians are
potentially reliable and efficient bioindicators (Welsh and Ollivier, 1998; Garg and Hippargi, 2007). They are very sensitive to even the slightest fluctuations in the environmental
settings (Cunnigham and Saigo, 1999).
Amphibians are a heterogeneous group of vertebrates with regard to their blood cell
count and size. However, the blood cell counts in Amphibia reported a wide individual
variation and considerable interspecies differences (Hutchison and Szarski, 1965; Szarski
and Czopek, 1966; Rouf, 1969; Sinha, 1983; Atatür et al., 1999; Cabagna et al., 2005) as
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138
Ç. Gül et. alii
well as in relation to body weight, age and sex (Arvy, 1947; Schermer, 1954; Goniaakowska, 1973; Sinha, 1983; Banerjee, 1988; Wojtaszek and Adamowicz, 2003), season (Zhukova
and Kubantsev, 1979; Sinha, 1983; Wojtaszek et al., 1997; Arserim and Mermer, 2008) and
altitudinal distribution (Arıkan, 1989; Ruiz et al., 1989).
Most studies on hematology in various species of Anura have dealt with blood cell
counts (Alder and Huber, 1923; Arvy, 1947; Kaplan, 1952; Stephan, 1954; Schermer, 1954;
Hutchison and Szarski, 1965; Arıkan, 1989) and cell sizes (Wintrobe, 1933; Foxon, 1964;
Hartman and Lessler, 1964; Szarski and Czopek, 1966; Kuramoto, 1981; Stöck and Grobe,
1997; Atatür et al., 1999; Arıkan et al., 2003; Cabagna et al., 2005; Gül and Tok, 2009).
Nevertheless, some hematological parameters of the anuran species, such as hematocrit
value (PCV), haemoglobin concentration (Hb), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and plasma total protein concentration, are very scarce and individual studies (Prosser and Weistein, 1950;
Haris, 1972; Carmena-Suero et al., 1980; Sinha, 1983; Ostejic et al., 2000; Wojtaszek and
Adamowicz, 2003; Coppo et al., 2005; Arserim and Mermer, 2008; Dönmez, 2009). So, the
possibilities of using quantitative and qualitative parameters of the blood in amphibians
undoubtedly hold great interest (Zhelev et al., 2006).
In the clinical investigation, blood samples are of great diagnostic value and can easily be obtained (Frye, 1991). The normal reference ranges of hematological parameters are
also important in assessing the status of the species population, and deviation from the
expected values can assess the impact of stress on the species (Dickinson et al., 2002).
Therefore, the objective of this study was to determine some hematological parameters [red blood cell count (RBC), white blood cell count (WBC), haemoglobin concentration (Hb), hematocrit value (PCV), mean cell volume (MCV), mean cell haemoglobin
(MCH), mean cell haemoglobin concentration (MCHC) and plasma total protein value]
in some Anuran species (Pelophylax ridibundus, Rana dalmatina, Pseudepidalea viridis,
Hyla arborea and Pelobates syriacus).
In addition, this study aims to investigate differences in the some hematological parameters of anuran species of terrestrial (P. viridis, P. syriacus and H. arborea), semi-aquatic
(R. dalmatina) and aquatic (P. ridibundus) nature.
MATERIAL AND METHODS
Specimens of the different Anuran species used in this study (P. viridis: n =10, H. arborea: n =
15, P. syriacus: n = 13, R. dalmatina: n = 15 and P. ridibundus: n = 10) were collected from their natural habitat and various localities of Canakkale, Turkey. Studies were carried out in the reproductive
period from February to April between 2008 and 2010. Blood samples of the live specimens were
obtained in the laboratory within one day of their capture. The blood samples were taken from the
etherized frogs by means of ventriculus punctures, via heparinized hematocrit capillaries (Arıkan et
al., 2003). The quantity of blood taken out from each specimens were 0.15 ml.
The blood cell counts were performed utilizing a Neubauer hemocytometer, and the standard
Hayem’s solution was used as diluting solutions for erythrocytes by Thoma pipettes, while for the
leukocytes, the method of Jerrett and Mays (1973) (which is a slight modification of Blain’s methodSturkie, 1954) was utilized; i.e. a 1:1 mix of neutral red diluted to 1/5000 with 0.07% physiological
saline and 12% formaline prepared with 0.07% physiological saline was used.
Haematology of Turkish Anurans
139
Blood from each frog or toad was placed into heparinised hematocrit capillaries and used to
determine the hematological parameters. Hematocrit value (PCV) was determined by the microhematocrit method. The tubes were then spun in a micro-hematocrit centrifuge for 5 min at 12000
rpm, and the hematocrit value (PCV) was calculated with a hematocrit reader. Hemoglobin concentration (Hb) was measured with a Sahli’s hemometer. The mean cell volume (MCV), mean cell
hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated mathematically from the above results. MCV was calculated by dividing hematocrit per liter of blood by total
RBC count (Tanyer, 1985). The blood samples were centrifuged at 3000 rpm for 10 min and it was
ensured that the plasma section be isolated from the blood cells. The plasma total protein quantity
in the plasma was measured using a Refractometer. Non-parametric tests and descriptive statistics
were calculated using SPSS (v10.0). Mann Whitney U test was applied between males and females.
RESULTS
Hematological parameters investigated in the present study are represented in Table 1.
There were some significant differences in some parameters when comparisons were made
between male and female data for one species. Differences between males and females in
terms of hemoglobin, hematocrit and mean cell volume in P. viridis were statistically significant (P < 0.05).
When the hematological values of anuran species were examined, the highest RBC
count, WBC count, hemoglobin concentration and hematocrit value were found in
Pseudepidalea viridis, whereas the lowest RBC count, WBC count, hemoglobin concentration and hematocrit value were found in R. dalmatina. The mean cell volume was detected
to be the highest in H. arborea and the lowest in R. dalmatina. The mean cell hemoglobin
was found to be the highest in H. arborea and the lowest in P. ridibundus. The mean cell
hemoglobin concentration was found to be the highest in Pseudepidalea viridis and the
lowest in P. ridibundus. The plasma total protein was found to be the highest in P. viridis
and the lowest in P. ridibundus. The hematological values of anuran species are given in
detail in Table 1.
DISCUSSION
Several authors, who worked on different species of Anura, mentioned individual variations concerning both the RBC and WBC counts (Alder and Huber, 1923; Klieneberger, 1927; Schermer, 1954; Hutchison and Szarski, 1965; Cabagna et al., 2005; Chiesa et al.
2006). In the present study, significant differences were found between males and females
only in P. viridis specimens in terms of hemoglobin, hematocrit and mean cell volume.
The RBC count was higher in males than in females of species P. ridibundus, R. dalmatina, H. arborea and P. syriacus. Similar observations were found by Arvy (1947) in R. temporaria, where males (450000) showed a higher RBC count than females (330000). Even
Sinha (1983) stated a higher count in males (320000) than in females (250000) in R. esculenta. Wojtaszek and Adamowicz (2003) have reported a higher number of RBC in males
(340000) than in females (290000) of Bombina bombina. However, the RBC count of P.
F
M
T
WBC
F
M
T
Hb
F
M
T
PCV
M
F
T
MCV
M
F
T
MCH
M
F
T
MCHC M
F
T
PTP
M
F
T
RBC
Sex
Mean
SD
Range
n
Mean
SD
Pelobates syriacus
Range
n
6 937666 212943 720000-1300000 11 765909 191818 560000-1200000 11
4 975000 94339.81 840000-1040000 2 652500 31819.80 630000-675000 4
10 952600 168908 720000-1300000 13 748461 180444 560000-1200000 15
6
5900
1391.40
4500-8000
11 2600
1798.33
1200-7400
7
4
6775
1117.66
5700- 8200
2
3250
353.55
3000-3500
2
10 6250
1302.34
4500-8200
13 2700
1662.82
1200-7400
9
6
15.76
0.79
14.60-17.00
11 10.38
2.46
6.00-12.80
11
4
12.95
0.91
12.00-14.20
2
9.30
3.53
6.80-11.80
4
10 14.64
1.65
12.00-17.00
13 10.21
2.50
6.00-12.80
15
6
58.50
6.94
52.00-70.00
5
40.00
0.08
27.00-50.00
11
4
43.75
3.30
40.00-47.00
2
38.00
0.11
30.00-46.00
3
10 52.60
9.40
40.00-70.00
7
40.00
0.08
27.00-50.00
14
6 638.63
96.05
538.46-793.06
5 468.20
83.07
366.67-595.24 11
4 450.18
34.10
403.85-476.19
2 577.35
143.06
476.19-678.52
3
10 563.25
122.40
403.85-793.06
7 499.39
104.15
366.67-678.52 14
6 174.18
33.94
130.77-213.89 11 138.59
35.34
100.00-228.57 11
4 133.93
17.53
115.38-152.38
2 141.37
47.28
107.94-174.81
4
10 158.08
34.27
115.38-213.89 13 139.02
35.04
100.00-228.57 15
6
27.24
3.10
23.03-30.77
5
28.86
2.73
25.60-31.90
11
4
29.67
1.94
27.65-32.00
2
24.21
2.19
22.66-25.76
3
10 28.21
2.86
23.03-32.00
7
27.53
3.30
22.66-31.90
14
5
8.16
1.36
6.00-9.50
4
7.12
1.65
5.00-9.00
11
4
7.50
0.57
7.00-8.00
2
6.50
0.70
6.00-7.00
2
9
8.00
1.14
6.00-9.50
6
6.91
1.35
5.00-9.00
13
n
Pseudepidalea viridis
Terrestrial
733636
647500
710666
3602
2800
3602
12.21
11.82
12.11
50.18
45.00
49.07
758.10
878.72
783.94
186.89
194.08
188.81
24.68
26.19
25.00
7.47
7.25
7.44
Mean
261965
212818
245516
652.83
1131.37
604.40
1.42
1.22
1.34
8.64
10.00
8.81
285.86
437.86
308.21
77.06
48.48
68.97
2.91
6.87
3.77
1.56
0.35
1.43
SD
Range
340000-1290000
400000-920000
340000-1290000
2000-4900
2000-3600
3100-4900
10.00-13.80
10.40-13.40
10.00-13.80
40.00-67.00
35.00-55.00
35.00-67.00
456.59-1411.76
546.88-1375.00
456.59-1411.76
106.98-382.35
144.57-260.00
106.98-382.35
20.24-31.16
18.91-32.57
18.91-32.57
6.00-10.00
7.00-7.50
6.00-10.00
Hyla arborea
Table 1. Red blood cell counts (RBC) (N/µL), white blood cell count (WBC) (µL), haemoglobin concentration (Hb) (g/dL), hematocrit value (PCV) (%),
mean cell volume (MCV) (fL), mean cell haemoglobin (MCH) (pg/cell), mean cell haemoglobin concentration (MCHC) (%) and plasma total protein (PTP)
(g/L). F: female, M: male, T: total; n: number of specimens, SD: standard deviation.
140
Ç. Gül et. alii
n
9
6
15
2
4
6
9
6
15
9
6
15
9
6
15
9
6
15
9
6
15
4
2
6
Sex
F
M
T
WBC
F
M
T
Hb
F
M
T
PCV
M
F
T
MCV
M
F
T
MCH
M
F
T
MCHC M
F
T
PTP
M
F
T
RBC
Mean
Range
n
5
5
10
3
2
5
5
5
10
5
5
10
5
5
10
5
5
10
5
5
10
3
3
6
SD
Range
886000 82945.76 780000-980000
762000 206567 650000-1130000
824000 162152 650000-1130000
3106
1462.20
1600-4520
2550
1484.92
1500-3600
2884
1308.92
1500-4520
9.52
1.47
8.20-12.00
8.66
1.94
7.40-12.10
9.09
1.68
7.40-12.10
43.19
4.12
39.45-48.78
38.19
13.48
23.52-59.25
40.69
9.76
23.52-59.25
488.83
38.74
428.80-524.52
499.71
105.91
336.00-621.79
494.27
75.40
336.00-621.79
107.50
13.28
95.65-129.03
114.69
8.07
105.71-124.24
111.09
11.03
95.65-129.03
21.95
1.66
20.39-24.60
23.78
5.11
18.72-31.46
22.87
3.71
18.72-31.46
5.50
0.50
5.00-6.00
5.83
0.76
5.00-6.50
5.66
0.60
5.00-6.50
Mean
Pelophylax ridibundus
Rana dalmatina
SD
Aquatic
Semiaquatic
716660 150997 550000-1020000
648330 84241.71 490000-720000
689330 129475 490000-1020000
2700
989.94
2000-3400
2675
960.46
2000-4100
2683
865.83
2000-4100
8.51
1.29
6.80-10.60
8.20
1.21
6.60-10.10
8.38
1.23
6.60-10.60
34.91
6.49
28.57-47.05
30.26
2.64
26.80-33.30
33.05
5.67
26.80-47.05
495.14
80.99
399.41-603.20
472.77
67.83
402.39-591.83
486.19
74.30
399.41-603.20
120.58
15.27
103.92-149.15
129.09
30.18
92.95-181.63
123.98
21.84
92.95-181.63
24.61
2.36
19.97-27.00
27.16
3.76
22.20-30.68
25.63
3.14
19.97-30.68
5.75
5.75
5.00-6.00
5.50
0.70
5.00-6.00
5.66
0.51
5.00-6.00
Table 1. Continued.
Haematology of Turkish Anurans
141
142
Ç. Gül et. alii
viridis was higher in females than in males. Mahapatra et al. (2010) have reported a higher RBC count in females (530000) than in males (480000) of the Polypedates maculatus.
A similar observation was found by Kaplan (1951, 1952) in Rana pipiens and by Arserim
and Mermer (2008) in R. macrocnemis. Meanwhile, no sexual dimorphism was reported
for the RBC count of R. pipiens (Rouf, 1969), R. catesbeiana and R. calamitans (Hutchison
and Szarski 1965) (Table 2).
The WBC counts vary depending on Anuran species, season, sex, nutritional conditions and some physiological conditions, such as diseases and breeding (Rouf, 1969;
Arıkan, 1989; Wojtaszek and Adamowicz, 2003). In the present study, significant differences were not found between males and females in all species in terms of WBC count.
The WBC counts were higher in males than in females of species P. ridibundus, R. dalmatina and H. arborea. A similar observation was found by Kaplan (1951, 1952) in R. pipiens
(16,134 in males; 14,134 in females) and by Wojtaszek and Adamowicz (2003) in B. bombina (9,734 in males; 7030 in females). However, they were higher in females than in males
of other species (P. syriacus and P. viridis). Arserim and Mermer (2008) put forth that the
WBC count in R. macrocnemis is higher in females (3613) than in males (3445, Table 2).
The haemoglobin concentration (Hb) and hematocrit values (PCV) were found to significantly differ between males and females in only P. viridis. Similar observations were
found by Sinha (1983) in R. esculenta, by Wojtaszek and Adamowicz (2003) in B. bombina, by Arserim and Mermer (2008) in R. macrocnemis and by Dönmez et al. (2009) in
Bufo bufo. Kaplan (1954) found statistically significant sexual differences in the hematocrit values of R. pipiens. The hemoglobin concentration of P. maculatus was higher in
females (7.80 g/100 ml) than in males (6.56 g/100 ml) (Mahapatra et al., 2010). Arserim
and Mermer (2008) put forth that the hematocrit values in Rana macrocnemis are higher
in females (35.00) than in males (32.00). Also, a similar observation was found by Haris
(1972) (Tables 1 and 2).
The values of MCV were found to significantly differ between males and females in
only P. viridis. In other species, there were no significant differences between males and
females. Generally, the MCV values were higher in females than in males of H. arborea, P.
syriacus and P. ridibundus. Similar MCV observations were found by Dönmez et al. (2009)
in B. bufo. Sinha (1983) and Arserim and Mermer (2008) reported that the MCV value
in R. esculenta and R. macrocnemis is higher in females than in males. MCH values were
higher in females than in males of P. syriacus, H. arborea, R. dalmatina and P. ridibundus. Sinha (1983) reported that the MCH value in R. esculenta is higher in males than in
females. MCHC values were higher in females than in males of P. viridis, H. arborea, R.
dalmatina and R. ridibunda. Mahapatra et al. (2010) has also reported a higher MCHC
value in females than in males of P. maculatus. However, Dönmez et al. (2009) stated a
higher MCHC value in males than in females of B. bufo (Tables 1 and 2).
Plasma total protein values were higher in males than in females of P. viridis, P. syriacus and H. arborea (Table 1).
Atatür et al. (1999) determined differences in the size of erythrocytes from several
species of Anura in Turkey and looked for the reasons in the different environmental conditions of the biotopes. Therefore, several researchers (Atatür et al., 1999; Zhelev et al.,
2006) have found that aquatic anurans have larger erythrocytes than semi-aquatic and terrestrial species.
Dönmez et al. (2009)
Bufo bufo
Gül and Tok (2009)
Rana ridibunda
Rana dalmatina
Bufo viridis
Bufo bufo
Hyla arborea
Pelobates syriacus
Mahapatra et al. (2010)
Polypedates maculatus
Arserim and Mermer (2008)
Rana macrocnemis
Alder and Huber (1923)
Hyla arborea
Arvy (1947)
Rana temporaria
Rouf (1969)
Rana pipiens
Harris (1972)
Rana pipiens
Carmena-Suero et al. (1980)
Hyla septentrionalis
Rana catesbeiana
Sinha (1983)
Rana esculenta
Arıkan et al. (1989)
Rana ridibunda
Ostojic et al. (2000)
Bufo spinulosus limensis
Arıkan et al. (2003)
Pelodytes caucasicus
Wojtaszek and Adamowicz (2003)
Bombina bombina
Coppo et al. (2005)
Rana catesbeiana
320000
250000
M
F
320500 (200000-650000)
2536 (800-4520)
415750 (310000-550000) 2683 (2000-4100)
721750 (450000-900000)
1646 (900-2500)
534722 (453000-703000) 2325 (1100-3800)
579583 (405000-703000)
2980 (460-4900)
657100 (543000-793000)
1216 (600-2500)
480000 (370000-580000) 14628 (12400-16200)
570000 (400000-710000) 16642 (14000-20000)
M
F
3445 (2600-4200)
3613 (2800-5200)
3527 (2600-5200)
506250 (280000-940000)
524000 (320000-900000)
514839 (280000-940000)
900000 (880000-920000)
870000 (840000-900000)
M
F
T
M
F
9734
7030
340000 (190000-465000)
290000 (240000-355000)
2560
3142
9600-38000
7850-25150
29000
WBC
M
F
776000
634000
326620
160000-540000
110000-450000
319400
450000
330000
674000
RBC
M
F
M
F
Sex
6.56 (5.20-8.40)
7.80 (6.40-9.00)
8.12 (5.60-12.10)
8.07 (6.20-11.00)
8.10 (5.60-12.10)
11.80 (11.20-12.40)
10.10 (9.40-10.80)
7.44 (4.99-12.20)
6.78 (3.38-8.31)
40.40
21.80
19.80
9.50
7.20
5.80
28.65 (18.51-37.60)
23.80 (16.00-32.07)
32.00 (19.00-42.00)
35.00 (16.00-46.00)
34.00 (16.00-46.00)
42.36 (41.53-43.20)
33.08 (28.57-37.60)
30.10 (25.00-39.00)
13.70-26.20
12.00-23.30
39.53
22.40
6.20
24.65
PCV
8.00-41.50
17.00-47.50
6.75
Hb
2.00-9.50
2.90-12.70
Table 2. The some hematological parameters in different Anura species refered by various authors.
Haematology of Turkish Anurans
143
Dönmez et al. (2009)
Bufo bufo
Gül and Tok (2009)
Rana ridibunda
Rana dalmatina
Bufo viridis
Bufo bufo
Hyla arborea
Pelobates syriacus
Mahapatra et al. (2010)
Polypedates maculatus
Arserim and Mermer (2008)
Rana macrocnemis
Alder and Huber (1923)
Hyla arborea
Arvy (1947)
Rana temporaria
Rouf (1969)
Rana pipiens
Harris (1972)
Rana pipiens
Carmena-Suero et al. (1980)
Hyla septentrionalis
Rana catesbeiana
Sinha (1983)
Rana esculenta
Arıkan et al. (1989)
Rana ridibunda
Ostojic et al. (2000)
Bufo spinulosus limensis
Arıkan et al. (2003)
Pelodytes caucasicus
Wojtaszek and Adamowicz (2003)
Bombina bombina
Coppo et al. (2005)
Rana catesbeiana
Table 2. Continued.
582.01 (474.57-700.00)
419.56 (360.57-462.16)
M
F
137.51 (119.29-146.66)
135.82 (125.00-160.00)
170.48 (91.49-229.41)
165.10 (87.78-257.89)
167.88 (87.78-257.89)
131.02 (127.27-134.78)
115.95 (111.90-120.00)
157.00 (121.00-197.00)
709.00 (505.00-788.00)
674.07 (425.53-1000)
716.39 (420.45-1105.26)
694.54 (420.45-1105.26)
470.74 (469.56-471.93)
378.90 (340.12-417.70)
158.30-268.10
145.30- 320.30
172.65
621.90
411.70-757.70
363.30-916.60
246.00
250.50
MCH
707.00
840.00
MCV
M
F
T
M
F
M
F
M
F
M
F
M
F
Sex
26.05 (20.57-32.34)
33.49 (28.06-40.00)
27.83 (26.97-28.70)
30.81 (28.72-32.90)
23.30 (20.20-31.40)
290.60-554.00
189.00-604.10
28.06
23.50
33.00
29.50
27.70
MCHC
4.34 (3.05-5.65)
PTP
144
Ç. Gül et. alii
Haematology of Turkish Anurans
145
In this study, the RBC counts were higher in terrestrial (P. viridis, P. syriacus and H.
arborea) than in semi-aquatic (R. dalmatina) species. There were variations in WBC count
of all species. Hemoglobin concentration, hematocrit value, MCV and MHC were higher
in terrestrial (P. viridis, H. arborea and P. syriacus) than in semi-aquatic (R. dalmatina)
and aquatic (P. ridibundus) species. The MCHC values were all similar to each other. The
plasma total protein was higher in terrestrial species than in semi-aquatic species.
In summary, it was reported that blood cell counts and sizes in anuran species displayed considerable individual variations and interspecies differences. Furthermore, in
this study, individual and interspecies variations were observed in the some hematological
parameters examined on the anuran species.
Acknowledgements
This study was supported by the Scientific Research Foundation of Çanakkale Onsekiz Mart
University under Project no: 2010/95. We would like to extend our gratitude to the foundation.
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